Changes In Cellular Redox Status Research Articles

S-Nitrosylation of AtSABP3 Antagonizes the Expression of Plant Immunity

Changes in cellular redox status are a well established response across phyla following pathogen challenge. In this context, the synthesis of nitric oxide (NO) is a conspicuous feature of plants responding to attempted microbial infection and this redox-based regulator underpins the development of plant immunity. However, the associated molecular mechanism(s) have not been defined. Here we show that NO accretion during the nitrosative burst promotes increasing S-nitrosylation of the Arabidopsis thaliana salicylic acid-binding protein 3 (AtSABP3) at cysteine (Cys) 280, suppressing both binding of the immune activator, salicylic acid (SA), and the carbonic anhydrase (CA) activity of this protein. The CA function of AtSABP3 is required for the expression of resistance in the host against attempted pathogen infection. Therefore, inhibition of AtSBAP3 CA function by S-nitrosylation could contribute to a negative feedback loop that modulates the plant defense response. Thus, AtSABP3 is one of the first targets for S-nitrosylation in plants for which the biological function of this redox-based post-translational modification has been uncovered. These data provide a molecular connection between the changes in NO levels triggered by attempted pathogen infection and the expression of disease resistance.

Effects of Glutathione Depletion on Hypoxia-induced Erythropoietin Production in Rats

In vitro studies have indicated that reactive oxygen species modifying cellular redox status are involved in hypoxia-induced erythropoietin (EPO) production. However, the effects of redox balance on hypoxia-induced EPO production in vivo are still not fully understood. To investigate the effect of the change in cellular redox status on EPO generation, we determined whether glutathione (GSH) depletion has a significant influence on hypoxia-induced EPO production in rats. For the inhibition of GSH synthesis, DL-buthionine-[S,R]-sulfoximine (BSO) was employed by intraperitoneal injection. Twenty male rats were assigned to one of four experimental groups: (1) normoxic placebo, (2) normoxic BSO, (3) hypoxic placebo, and (4) hypoxic BSO. Hypoxic groups were exposed to a simulated normobaric hypoxic condition (4,500 m above sea level) for 12 hours. BSO treatment resulted in a significant depletion of total GSH levels in kidney and plasma in both conditions. However, the hypoxia-induced elevation in serum EPO concentration was not completely affected by the inhibition of GSH synthesis. These data demonstrate that GSH depletion in the kidney is not involved in the increase in serum EPO concentration in response to systemic hypoxia. It is also conceivable that the cellular redox changes could not function as a primary regulator of hypoxia-induced renal erythropoietin formation in vivo.

Superoxide dismutase and glutathione reductase overexpression in wheat protoplast: photooxidative stress tolerance and changes in cellular redox state

In previous works, we have established a correlation between antioxidant system response and tolerance to drought, osmotic stress and photooxidative stress of different wheat cultivars with contrasting drought tolerance. In the present work, a protocol to obtain and transform wheat protoplasts was established. Transgenic protoplasts with Manganese Superoxide Dismutase (Mn-SOD) (E.C.: 1.15.1.1) and Glutathione Reductase (GR) (E.C.: 1.6.4.2) overexpression in chloroplasts were obtained, and their responses to photooxidative stress were characterized. Protoplasts with Mn-SOD or GR overexpression, showed different responses and tolerance to photooxidative stress. Protoplasts with Mn-SOD overexpression showed lower levels of oxidative damage, higher level of endogenous hydrogen peroxide and a great induction of total SOD and GR activities during photooxidative treatments. In protoplasts with GR overexpression the oxidative damage provoked by the photooxidative treatment was similar to control protoplasts, the GSH content and GSH/GSH + GSSG ratio were higher than control and Mn-SOD transformed protoplast, and total SOD and GR activities were not induced. Our results suggest that the differential responses and tolerance to photooxidative stress given by Mn-SOD or GR overexpression, also depend on the effects of these enzyme activities over the cellular redox state balance, which modulate the responses to photooxidative stress.

Current status and future prospects in the search for protein biomarkers of immunosenescence

Complex adaptations including changes in cellular redox status, the production of high levels of pro-inflammatory cytokines and alterations in immunity occur as the result of aging of the immune system (immunosenescence). These events are thought to underlie the progression of chronic degenerative diseases of aging, such as atherosclerosis, Type 2 diabetes and Alzheimer’s disease. It is envisaged that identifying early biomarkers of immune aging would aid in identifying individuals at risk of age-related disease and would allow the discovery of novel intervention strategies. Proteomics has emerged as a rapidly expanding and innovative field, investigating protein expression, interaction and function at a global level. Several proteomic strategies, including use of mass spectrometry and non-mass spectrometry-based detection systems (including secondary antibody labeling with fluorescent tags) may be particularly advantageous in identifying biomarkers of immune health. Application of these approaches may identify factors that both contribute to (and define) age-dependent deregulation of the immune system.

Redox signalling in cardiovascular disease

Oxidative stress has almost universally and unequivocally been implicated in the pathogenesis of all major diseases, including those of the cardiovascular system. Oxidative stress in cells and cardiovascular biology was once considered only in terms of injury, disease and dysfunction. However, it is now appreciated that oxidants are also produced in healthy tissues, and they function as signalling molecules transmitting information throughout the cell. Conversely, when cells move to a more reduced state, as can occur when oxygen is limiting, this can also result in alterations in the function of biomolecules and subsequently cells. At the centre of this 'redox signalling' are oxidoreductive chemical reactions involving oxidants or reductants post translationally modifying proteins. These structural alterations allow changes in cellular redox state to be coupled to alterations in cell function. In this review, we consider aspects of redox signalling in the cardiovascular system, focusing on the molecular basis of redox sensing by proteins and the array of post-translational oxidative modifications that can occur. In addition, we discuss studies utilising proteomic methods to identify redox-sensitive cardiac proteins, as well as those using this technology more broadly to assess redox signalling in cardiovascular disease.

Effects of ginsenosides Re and Rg3 on intracellular redox state and cell proliferation in C6 glioma cells

BackgroundCellular redox state is important to cell growth and death. The growth of tumor cells may be modulated by intracellular reduced glutathione/oxidized glutathione (GSH/GSSG). The present study aims to investigate the effects of ginsenosides Re and Rg3 on cellular redox state and cell proliferation in C6 glioma cells.MethodsCultured C6 glioma cells were exposed to various concentrations of either Rg3 or Re for 24 hours. Cell growth and death were measured by the BrdU incorporation assay and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay respectively. Cellular redox state was determined by free radical production using flow cytometry and GSH/GSSG using spectrofluorometry.ResultsAt a sub-lethal concentration, Re suppressed cell proliferation with a significant decrease in BrdU incorporation. Re did not increase reactive oxygen species (ROS) production but increased GSH/GSSG via increased activity of gamma glutamylcystenyl synthase (γ-GCS). In contrast, Rg3 increased free radical production and reduced GSH/GSSG. The effects of Rg3 were probably due to increased activity of glutathione peroxidase (GPx).ConclusionRe and Rg3 alter cellular redox state of C6 glioma cells in opposite directions. Changes in cellular redox state induced by Re and Rg3 are correlated with the proliferation rates of C6 glioma cells.

Redox Control of Calcium Channels: From Mechanisms to Therapeutic Opportunities

Calcium plays an integral role in cellular function. It is a well-recognized second messenger necessary for signaling cellular responses, but in excessive amounts can be deleterious to function, causing cell death. The main route by which calcium enters the cytoplasm is either from the extracellular compartment or internal addistores via calcium channels. There is good evidence that calcium channels can respond to pharmacological compounds that reduce or oxidize thiol groups on the channel protein. In addition, reactive oxygen species such as hydrogen peroxide and superoxide that can mediate oxidative pathology also mediate changes in channel function via alterations of thiol groups. This review looks at the structure and function of calcium channels, the evidence that changes in cellular redox state mediate changes in channel function, and the role of redox modification of channels in disease processes. Understanding how redox modification of the channel protein alters channel structure and function is providing leads for the design of therapeutic interventions that target oxidative stress responses.

Structure of the Redox Sensor Domain of Azotobacter vinelandii NifL at Atomic Resolution: Signaling, Dimerization, and Mechanism,

NifL is a multidomain sensor protein responsible for the transcriptional regulation of genes involved in response to changes in cellular redox state and ADP concentration. Cellular redox is monitored by the N-terminal PAS domain of NifL which contains an FAD cofactor. Flavin-based PAS domains of this type have also been referred to as LOV domains. To explore the mechanism of signal recognition and transduction in NifL, we determined the crystal structure of the FAD-bound PAS domain of NifL from Azotobacter vinelandii to 1.04 A resolution. The structure reveals a novel cavity within the PAS domain which contains two water molecules directly coordinated to the FAD. This cavity is connected to solvent by multiple access channels which may facilitate the oxidation of the FAD by molecular oxygen and the release of hydrogen peroxide. The structure contains a dimer of the NifL PAS domain that is structurally very similar to those described in other crystal structures of PAS domains and identifies a conserved dimerization motif. An N-terminal amphipathic helix constitutes part of the dimerization interface, and similar N-terminal helices are identified in other PAS domain proteins. The structure suggests a model for redox-mediated signaling in which a conformational change is initiated by redox-dependent changes in protonation at the N5 atom of FAD that lead to reorganization of hydrogen bonds within the flavin binding pocket. A structural signal is subsequently transmitted to the beta-sheet interface between the monomers of the PAS domain.

Preclinical Perspectives on Garlic and Cancer

ABSTRACT Evidence continues to point to the anticancer properties of fresh garlic extracts, aged garlic, garlic oil, and a number of specific organosulfur compounds generated by processing garlic. These anticarcinogenic and antitumorigenic characteristics appear to arise through both dose- and temporal-related changes in a number of cellular events involved with the cancer process, including those involving drug metabolism, immunocompetence, cell cycle regulation, apoptosis, and angiogenesis. The ability of garlic and related allyl sulfur compounds to block tumors in the colon, lung, breast, and liver suggests general mechanisms that are not tissue specific. Whereas relatively few studies have compared the relative efficacy of water- and lipid-soluble allyl sulfur compounds, those that have when using chemically induced carcinogen models suggest little difference in response, whereas tumor proliferation/apoptosis is highly dependent on the species provided. A shift in sulfhydryl groups, alterations in glutathione:oxidized glutathione ratios, and resultant changes in cellular redox status may be involved in some of the phenotypic changes caused by allyl sulfur compounds. Such changes in thiols by allyl sulfurs may also account for the observed hyperphosphorylation of specific cell cycle proteins and the histone hyperacetylation that has been correlated with suppressed tumor cell proliferation. Whereas the anticarcinogenic and antitumorigenic data to date are impressive, additional studies are needed with more modest exposure to allyl sulfur compounds over prolonged periods. Likewise, additional studies are needed that incorporate transgenic and knockout models to assist in the identification of molecular targets for garlic and its associated allyl sulfur components.

Preclinical Perspectives on Garlic and Cancer

Evidence continues to point to the anticancer properties of fresh garlic extracts, aged garlic, garlic oil, and a number of specific organosulfur compounds generated by processing garlic. These anticarcinogenic and antitumorigenic characteristics appear to arise through both dose- and temporal-related changes in a number of cellular events involved with the cancer process, including those involving drug metabolism, immunocompetence, cell cycle regulation, apoptosis, and angiogenesis. The ability of garlic and related allyl sulfur compounds to block tumors in the colon, lung, breast, and liver suggests general mechanisms that are not tissue specific. Whereas relatively few studies have compared the relative efficacy of water- and lipid-soluble allyl sulfur compounds, those that have when using chemically induced carcinogen models suggest little difference in response, whereas tumor proliferation/apoptosis is highly dependent on the species provided. A shift in sulfhydryl groups, alterations in glutathione:oxidized glutathione ratios, and resultant changes in cellular redox status may be involved in some of the phenotypic changes caused by allyl sulfur compounds. Such changes in thiols by allyl sulfurs may also account for the observed hyperphosphorylation of specific cell cycle proteins and the histone hyperacetylation that has been correlated with suppressed tumor cell proliferation. Whereas the anticarcinogenic and antitumorigenic data to date are impressive, additional studies are needed with more modest exposure to allyl sulfur compounds over prolonged periods. Likewise, additional studies are needed that incorporate transgenic and knockout models to assist in the identification of molecular targets for garlic and its associated allyl sulfur components.

High levels of antioxidant enzymatic defence assure good protection against hypoxic stress in spontaneously diabetic rats

Recent data from literature report that reactive oxygen species (ROS) seem to play a crucial role in the etiology of both types I and II diabetes. This may render diabetic individuals more prone to oxidative injury when challenged with hypoxic stress. It is in fact well known that many diabetic complications cause ischaemic episodes, with a consequent reduction in oxygen supply to various tissues and organs. To check this hypothesis, in this work we tested type I diabetic individuals’ antioxidant capability towards a hypoxic-mediated oxidative challenge. In particular, spontaneously diabetic and age-matched non-diabetic biobreeding (BB) Wistar rats were submitted to chronic normobaric hypoxia, and the response of antioxidant enzymes, as well as redox-sensitive transcription factor NF-κB and p53, were monitored. Results show that diabetic subjects present a dramatic enhancement in the major antioxidant enzymes activities, thus supporting the notion of diabetes-related changes in cellular redox status. This allows diabetic individuals to counteract hypoxia-mediated oxidative challenge better than the non-diabetic counterpart. Also the behaviour of both the redox-sensitive nuclear transcription factor NF-κB and p53 protein in response to hypoxic stimulation seems to support the hypothesis of a better ROS scavenging efficiency in diabetics under hypoxic conditions. In conclusion, high levels of antioxidant enzymatic defences in diabetic BB rats reflect a positive adaptive response able to assure an efficient protection not only against chronic, diabetes-mediated reactive oxygen species (ROS) overproduction, but also versus further oxidative damage.

Oxygen‐sensing pathway for SK channels in the ovine adrenal medulla

1. The intracellular pathways that modulate the opening of oxygen-sensitive ion channels during periods of hypoxia are poorly understood. Different tissues appear to use either NADPH oxidase or a rotenone-sensitive mechanism as an oxygen sensor. The aim of the present study was to identify the oxygen-sensing pathway in the oxygen-sensitive sheep adrenal medullary chromaffin cell (AMCC). 2. The whole-cell patch-clamp technique was used to measure K+ currents in dissociated adult ovine chromaffin cells as well as SK channel currents expressed in the H4IIE cell line. 3. Diphenyliodonium, an inhibitor of NADPH oxidase, had no effect on the hypoxia-evoked closure of K+ channels in primary AMCC, whereas the mitochondrial inhibitor rotenone abolished the hypoxia-evoked response. Both these compounds significantly reduced K+ current amplitude under normoxic conditions. 4. One possible mechanism through which the oxygen sensor may modulate K+ channel activity is by altering the redox state of the cell. In sheep AMCC, altering the redox state by the addition of H2O2 to the extracellular solution increased K+ conductance. 5. The oxygen-sensitive K+ (Ko2) channels in sheep chromaffin cells are from the SK family and the whole-cell conductance of cells expressing mouse SK2 or SK3, but not human SK1, was increased by H2O2 and decreased by the reducing agent dithiothreitol. 6. These studies show that, in sheep AMCC, Ko2 channels are modulated via a rotenone-sensitive mechanism and that alteration of the cellular redox state mimics the change produced by alterations in Po2. In a heterologous expression system, SK2 and SK3 channels, the channels that initiate hypoxia-evoked changes in AMCC function, are modulated appropriately by changes in cellular redox state.

Redox Regulation of the Cdc25 Phosphatases

The Cdc25 phosphatases are essential for cell-cycle control in eukaryotes under normal conditions and in response to DNA damage via checkpoint controls. Recent evidence indicates direct control of the Cdc25s, and therefore the cell cycle, in response to changes in cellular redox status. These redox changes may originate intracellularly from mitochondrial leakage or in response to specific external triggers leading to production of reactive oxygen species (ROS). This review shows that the known chemistry and biology of the Cdc25s favor a direct role for these phosphatases in temporarily blocking cell-cycle progression until favorable reducing conditions are restored. First, the Cdc25s contain a highly reactive cysteine at the active site that can react directly with ROS, leading to enzyme inactivation. Second, the ROS-inactivated form of Cdc25 is expected to prevent cell-cycle progression based on precedent from cellular responses to DNA damage. Third, ROS-mediated oxidation of the Cdc25s leads to an intramolecular disulfide that is readily reversible by the cellular reductant thioredoxin. Finally, in vivo data supporting a direct role for the Cdc25s in redox regulation are considered.

Selective expression of glutathione S-transferase genes in the murine gastrointestinal tract in response to dietary organosulfur compounds.

A short-term feeding regimen was designed to analyze the effects of compounds such as diallyl disulfide (DADS), diallylthiosulfinate (allicin) from garlic and butylated hydroxyanisole (BHA) on glutathione S-transferase (GST) expression in the gastrointestinal tract and liver of male mice. After animals were force-fed these compounds, tissue GSTs were purified and individual subunits resolved by HPLC and identified on the basis of mass spectrometry (ESI MS) and immunoreactivity data. The effects of DADS and allicin on GST expression were especially prominent in stomach and small intestine, where there were major coordinate changes in GST subunit profiles. In particular, the transcripts of the mGSTM1 and mGSTM4 genes, which share large segments of common 5'-flanking sequences, and their corresponding subunits were selectively induced. Levels of alpha class subunits also increased, whereas mGSTM3 and mGSTP1 were not affected. The inducible mGSTA5 and non-responsive mGSTM3 subunits had not been identified previously. Liver and colon GSTs were also affected to a lesser extent, but this short-term feeding regimen had no effect on GST subunit patterns from other organs, including heart, brain and testis. Real-time PCR (TaqMan) methods were used for quantitative estimations of relative amounts of the mRNAs encoding the GSTs. Effects on the transcripts generally paralleled changes at the protein level, for the most part, however, the greatest relative increases were observed for those mRNAs that were expressed at low abundance constituitively. Mechanisms by which the organosulfur compounds operate to affect GST transcription could involve reversible modification of certain protein sulfhydryl groups, shifts in reduced glutathione/oxidized glutathione ratios and resultant changes in cellular redox status.

Redox regulation of reactive oxygen species-induced p38 MAP kinase activation and barrier dysfunction in lung microvascular endothelial cells.

Reactive oxygen species (ROS)-mediated compromise of endothelial barrier integrity has been implicated in a number of pulmonary disorders, including adult respiratory distress syndrome, pulmonary edema, and vasculitis. The mechanisms by which ROS increase endothelial permeability are unclear. We hypothesized that ROS-induced changes in cellular redox status (thiols) may contribute to endothelial barrier dysfunction. To test this hypothesis, we used N-acetylcysteine (NAC) and diamide to modulate intracellular levels of cellular glutathione (GSH) and investigated hydrogen peroxide (H(2)O(2))-mediated mitogen-activated protein kinase (MAPK) activation and transendothelial electrical resistance (TER). Exposure of bovine lung microvascular endothelial cells (BLMVECs) to H(2)O(2), in a dose- and time-dependent fashion, increased endothelial permeability. Pretreatment of BLMVECs with NAC (5 mM) for 1 h resulted in partial attenuation of H(2)O(2)-induced TER (a measure of increase in permeability) and GSH. Furthermore, treatment of BLMVECs with diamide, which is known to reduce the intracellular GSH, resulted in significant reduction in TER, which was prevented by NAC. To understand further the role of MAPKs in ROS-induced barrier dysfunction, we examined the role of extracellular signal-regulated kinase (ERK) and p38 MAPK on H(2)O(2)- and diamide-mediated permeability changes. Both H(2)O(2) and diamide, in a dose-dependent manner, activated ERK and p38 MAPK in BLMVECs. However, SB203580, an inhibitor of p38 MAPK, but not PD98059, blocked H(2)O(2)- and diamide-induced TER. Also, NAC prevented H(2)O(2)- and diamide-induced p38 MAPK, but not ERK activation. These results suggest a role for redox regulation of p38 MAPK in ROS-dependent endothelial barrier dysfunction.

Redox-dependent downregulation of Rho by Rac.

Rac and Rho GTPases function as critical regulators of actin cytoskeleton remodelling during cell spreading and migration. Here we demonstrate that Rac-mediated reactive oxygen species (ROS) production results in the downregulation of Rho activity. The redox-dependent decrease in Rho activity is required for Rac-induced formation of membrane ruffles and integrin-mediated cell spreading. The pathway linking generation of ROS to downregulation of Rho involves inhibition of the low-molecular-weight protein tyrosine phosphatase (LMW-PTP) and then an increase in the tyrosine phosphorylation and activation of its target, p190Rho-GAP. Our findings define a novel mechanism for the coupling of changes in cellular redox state to the control of actin cytoskeleton rearrangements by Rho GTPases.

Redox regulation and storage processes during maturation in kernels of Triticum durum.

Metabolic changes during the development and maturation of Triticum durum Desf. (L.) kernels were studied, with particular emphasis on changes in the redox state of ascorbate and glutathione, as well as in the activities of the enzymes responsible for the recycling of their oxidized forms (ascorbic free radical reductase, EC 1.6.5.4; dehydroascorbate reductase, EC 1.8.5.1; glutathione reductase, EC 1.6.4.2) and for detoxification or utilization of hydrogen peroxide (ascorbate peroxidase, EC 1.11.1.11; catalase, EC 1.11.1.6). In parallel with this analysis, the production and storage of reserve compounds was studied, in particular, soluble carbohydrates (mono- di-saccharides and fructans) and the transition from sulphydryl groups to disulphide bridges into proteins. The results indicate that both the activities of the ascorbate and glutathione redox enzymes and that of catalase are high before the start of drying maturation, after which they decrease. Moreover, analysis of the redox state of ascorbate and glutathione pairs and the sulphydryl to disulphide transition into proteins suggests that these three parameters are tightly related during kernel maturation, thus confirming the involvement of the two redox pairs in protein maturation as well as in protection against reactive oxygen species. The physiological implications of the changes in cellular redox state and in soluble carbohydrates for the acquisition of desiccation tolerance and reaching the resting phase in orthodox seeds are also discussed.

Redox Regulation Gets More Complicated

Gene expression is regulated in response to changes in cellular redox status. Kim et al. provide evidence that not all agents that alter redox status are perceived as identical. Using biochemical analysis of the bacterial transcription factor OxyR, which has a cysteine redox center, the authors analyzed the accessibility of the six cysteines, the structural changes associated with cysteine modification, and finally the effect of various oxidants on DNA-binding and transcription activation. Only one cysteine (C 199 ) was accessible and reacted with peroxide to form OxyR-SOH, with S-nitrosoglutathione to form OxyR-SNO, or with glutathione disulfide to form OxyR-SSG. In vivo, OxyR was fully reduced under nonstimulated conditions. Peroxide treatment of the bacteria to oxidize OxyR led to the production of OxyR-SSG. In vitro transcription assays demonstrated that OxyR-SOH, OxyR-SNO, and OxyR-SSG stimulated transcription from both the katG and OxyS promoters. Each of the modified forms of OxyR exhibited different DNA-binding characteristics at the katG and OxyS promoters. However, the DNA-binding affinity did not correlate with transactivating potential. Cystine modification altered DNA-binding and transcriptional activation capability independently. These results provide an initial answer to how gene regulation can be tuned to the specific redox stress and suggest that the redox center in OxyR is not a simple on-off switch, but is a more selective and specific sensor for redox changes. S. O. Kim, K. Merchant, R. Nudelman, W. F. Beyer, Jr., T. Keng, J. DeAngelo, A. Hausladen, J. S. Stamler, OxyR: A molecular code for redox-related signaling. Cell 109 , 383-396 (2002). [Online Journal]

P53 and redox state in etoposide-induced acute myeloblastic leukemia cell death

We investigated whether p53, being a redox-sensitive protein, has a role in the responsiveness of AML cells to etoposide. Two subclones of the OCI/AML-2 cell line, the etoposide-sensitive (ES) and the etoposide-resistant (ER), were used as models. Sensitivity to etoposide was measured by trypan blue and annexin V assays. Etoposide-induced peroxide formation was associated with the induction of cell death. Evident expression of mutated p53 was observed in both subclones in basal growth conditions as analysed by Western blotting and flow cytometry. After etoposide exposure for up to 24 hours, some nuclear accumulation of p53 was observed in the ER subclone, as analysed by Western blotting. The conformation of p53, however, was not changed from mutated toward wild-type during exposure in either of the subclones as analysed by flow cytometry. In conclusion, etoposide-induced change in cellular redox state was associated with apoptosis, but was not a sufficient stimulus for p53 to make its conformation active. Thus, mutated p53 seems to have no role in etoposide-induced apoptosis.

Redox Around the Clock

Circadian rhythms are controlled by an evolutionarily conserved transcriptional feedback system whose activity fluctuates as a function of the 24-hour light-dark cycle. Extrinsic factors, such as changes in food intake, can advance or delay the circadian clock, but the mechanism by which this "entrainment" occurs remains unclear. A key regulator of circadian rhythms, Clock, is expressed in the suprachiasmatic nucleus (the brain region regarded as the master pacemaker) and regulates the expression of genes encoding other clock components. Reick et al. and Rutter et al. show that the NPAS2 transcription factor performs a function similar to Clock in the mammalian forebrain and that the DNA binding activity of both these transcription factors in vitro is regulated by the redox state of nicotinamide adenine dinucleotide (NAD) cofactors. Because changes in food intake are associated with changes in cellular redox state, the authors propose that redox control of Clock and NPAS2 activity may explain how food and other extrinsic factors entrain the molecular clock. A Perspective by Schibler et al. accompanies the two reports. M. Reick, J.A. Garcia, C. Dudley, S.L. McKnight, NPAS2: An analog of Clock operative in the mammalian forebrain. Science 293 , 506-509 (2001). [Abstract] [Full Text] J. Rutter, M. Reick, L.C. Wu, S.L. McKnight, Regulation of Clock and NPAS2 DNA binding by the redox state of NAD cofactors. Science 293 , 510-514 (2001). [Abstract] [Full Text] U. Schibler, J.A. Ripperger, S.A. Brown, Chronobiology--reducing time. Science 293: 437-438 (2001). [Full Text]